Nitration of Tyrosyl Residues in Human α‐Lactalbumin

Abstract

a-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8 -32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration.These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted cr-lactalbumin.Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of a-lactalbumin.Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human a-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50.Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives. Since the nitration of tryptophan residues was claimed to take place only when high molar excess of reagent over protein was employed, the use of C(NO;)4 as a chemical modifying agent appeared to retain much of its interest in studying which site of human cr-lactalbumin is interacting with human A protein in the lactose synthase system.Tyrosine side-chains were indeed likely candidates for playing a definite role in either the primary or even the secondary structure of a-lactalbumin. Align- ment of sequences for the three a-lactalbumins which have been completely worked out (bovine, guinea-pig and human; [6-81) indicates that each of the four tyrosines occupies the same position throughout. Furthermore, their immediate neighbours are also unchanged. Such is, however, not the case for kangoroo a-lactalbumin ([9] partial sequence from 1 -42) in which tyrosines-18 and 36 are replaced by, respectively, valine and leucine ; moreover, their neighbours are not conserved.From these results it seemed worthwhile to attempt a specific substitution of tyrosines in order to find out whether these residues are part of some site of association between a-lactalbumin and the A protein.The chemical modification of tyrosyl residues in bovine a-lactalbumin has already been reported. Reaction with C(N02)4 was used [lo-121 leading, however, to contradicto...

Application of the pH-jump method to the titration of tyrosine residues in bovine .alpha.-lactalbumin

“…One or two tyrosines in -lactalbumin were known to be unreactive with cyanuric fluoride or with iV-acetylimidazole, which may be consistent with the present results (Gorbunoff, 1967;Kronman et al, 1971). Prieels et al (1975) have suggested that Tyr-103 in human -lactalbumin, in which all the tyrosines occupy the same positions as in bovine a-lactalbumin, is more easily nitrated than Tyr-18 and that these two residues are more exposed than Tyr-36 and -50. The results are also consistent with the assignment in Table I.…”

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

“…Other possible modifications that were investigated included nitrotryptophane, 36 nitrophenylalanine, 37 hydroxyphenylalanine, 37 nitrohistidine, 35 and conversion of sulfhydryl to sulfenic and sulfonic acid in cysteine. 38 The mass spectral data did not show evidence for any of these modifications.…”

Requirements for heme and thiols for the nonenzymatic modification of nitrotyrosine

“…teins (43). Chemical nitration of functionally important tyrosine residues by tetranitromethane was often found to inactivate or alter the properties of the enzyme under investigation (44)(45)(46).…”